In HIV infected patients, plasma HIV load increases with concomitant opportunistic infections, including Mycobacterial and Chlamydial infections suggesting cell activation and active virus replication. The role of the innate immune system and Toll-like Receptors (TLRs) in HIV replication during opportunistic infections are unknown. I have shown that LPS activates HIV-long terminal repeat (LTR) through innate immune system receptor TLR4 via IL-1R signaling molecules. In preliminary experiments, I observed that TLR2 transduces mycobacterial antigen Soluble Tuberculosis Factor (STF) response to activate HIV-LTR, and that TLR4 (LPS) and TLR2 (STF) ligands induce synergistic activation of HIV-LTR. The molecular mechanisms involved in TLR2-mediated STF activation of HIV-LTR and the mechanisms of the synergy observed between TLR2 and TLR4 ligands for HIV-LTR transactivation are unknown. Furthermore, it is unknown whether Chlamydial effector molecule, Heat Shock Protein 60 (cHSP60) is recognized by TLR4 or TLR2. The overall goals of this proposal are to determine the role of the innate immune receptors, TLRs, and the molecular signaling mechanisms involved in mycobacterial and chlamydial antigen-induced HIV-1-LTR activation and replication, as well as the molecular mechanisms involved in the synergistic activation of HIV-LTR by TLR2 and TLR4 ligands. The hypothesis: I hypothesize that activation of the innate immune system through TLRs (TLR2 for STF, and TLR4 for LPS and cHSP60) mediate the bacterial and mycobacterial antigen-induced activation of NF-kB and HIV-LTR via IL-1R signaling molecules. I also hypothesize that TLR4 and TLR2 ligands synergistically induce HIV- LTR transactivation. Thus, TLRs may provide a potential molecular link between opportunistic infections such as Mycobacterial tuberculosis and Chlamydial infections and increased HIV replication. The specific aims are: (1)-To assess the role of TLR2 and TLR4, and to determine the functional cooperation and synergy of TLRs in Soluble Tuberculosis Factor (STF)-, and chlamydia HSP60 (cHSP60)-induced HIV-LTR activation in macrophages. (2)-To investigate the molecular signaling mechanisms involved in TLR20 and TLR4-induced HIV-LTR transactivation and HIV replication. (2a)-To determine the role of the IL-1 signaling molecules, Tollip, MyD88 and NF-kB activation in TLR2-mediated HIV-LTR activation. (2b)-To study the role of PI3 Kinase, Rac1 and Akt in TLR 2 and TLR4-induced activation of HIV- LTR and HIV replication. (2c)-To investigate the molecular mechanism of the synergy between TLR2 and TLR4 ligands forHIV-LTR activation. Significance: Improved understanding of the role of innate immune receptors, such as TLRs and the molecular signaling mechanisms involved in microbial antigen induced HIV-LTR transactivation and HIV replication may provide new targets to better control HIV replication during mycobacterial and chlamydial infections.